L-prolyl-l-seryl-l-lysyl-l-alanyl-l-phenylalanyl-l-isoleucyl-glycyl-l-leucyl-l-methionine amide



R. BOISSON NAS ET AL -L-ISOLEUCYL-GLYCYL-L-LEUCYL-L-METHIONINE AMIDE 2Sheets-Sheet l N z. E: .33 ...w m: d2 i -:z- E: zu o: 2^.. ai 4.95 Nrz--:i a@ iA #2-12-- w: mi |||s U mi .Ez so.. zw .E.

ATT RNEY June 27, 1967 Filed Dec.

June 27, 1967 n. BolssoNNAs ETAL 3,328,382

LPROLYL-L-SERYL-L-*LYSYL-IrALANYL-L-PHENYLALANYL'-1.1-ISOLEUCYL-GLYCYL-L-LEUCYL-IJMETHIONINE AMIDE Filed Dec. 2, 1963 2Sheets-Sheet 2 -OCH3 CTB Lys |-0CH3 FIG. 4

Ala Phe Ile Alu Phe Ile cracre-

CTB-

cTe-L Pro Ser ser

Pro Ser- Lys CTB CTB-l Pm H-Lprc INVENTORS ROGER BOISSONNAS ATTORNEYUnited States Patent O 3,328,382 L PROLYL L SERYL L LYSYL L ALANYL- LPHENYLALANYL L ISOLEUCYL GLYCYL- L LEUCYL L METHIQNINE AMIDE RogerBoissonnas, Bottmingen, and Edmond Sandrin, Basel, Switzerland,assignors to Sandoz Ltd., Basel, Switzerland Filed Dec. 2, 1963, Ser.No. 327,448 Claims priority, application Switzerland, Dec. 10, 1962,14,450/ 62 2 Claims. (Cl. 260--112.5)

The present invention relates to a hitherto unknown nonapeptide, itssalts,

The present invention provides L-prolyl-L-seryl-L-lysyl-L-alanyl-L-phenylalanyl-L-isoleucyl-glycyl-L leucyl L- methionine-amideand its acid addition salts. Suitable acids for acid addition saltformation are, for example, acetic, triuoroacetic, p toluene sulfonic,tartaric, gluconic, maleic, malic, methanesulfonic and citric acids.

The said nonapeptide is shown in FIGURE IV of the accompanyingdiagram-matic drawings.

The following abbreviations are used in the text and in the drawings:

H-Pro-OH-L-proline H-Ser-OH-L-serine H-Lys-OH-L-lysineH-Ala-OH-L-alanine H-Phc-OH-L- phenylalanine H-Ile-OH-L-isoleucineH-Gly-OH-glycine H-Leu-OH-L-leucine H-Met-NHZ-L-methionine .amideCBO-carbobenzoxy CTB-carbo-tert-butoxy Tri-trityl ONP-p-nitrophenyloxyOMe-methoxy OBz-benzyloxy The nonapeptide of the invention may beobtained by condensing L-prolyl-L-seryl-L-lysine, a new tripeptide, inwhich the e-amino radical of the lysine radical is protected by asuitable protective group, the a-amino radical of the proline radical bya similar or different protective group and if desired the carboxylradical of the lysine radical may be substituted by a radical which iscapable of reacting with amino radicals, with L-alanyl-L-phenylalanyl-L-isoleucyl-glycyl-L-leucyl-L-methionine-amide, a hexapeptide, andremoving the protective groupsV in one or more stages from the resultingL-prolyl-L-seryl-L-lysyl-L-alanyl-L-phenylalanyl-L-isoleucyl-glycyl-L-leucyl-L methionineamide of whichthe e-amino radical of the lysine radical and the terminal a-aminoradical of the proline radical are each protected by a suitableprotective group.

Examples of suitable protective groups for the terminal a-amino radicalof the proline radical are the carbobenzoxy, toluenesulfonyl,carbo-tert-butoxy, formyl, trifluoroacetyl and p-nitrocarbobenzoxyradicals. Examples of protective groups for the e-amino radical of thelysine radical are the carbobenzoxy, toluenesulfonyl, carbo-tertbutoxyphthalyl, formyl, trifluoroacetyl and p-nitrocarbobenzoxy radicals.Examples of groups for the substitution of the carboxyl radical of thelysine radical capable of reacting with the terminal amino radical ofthe hexapepice tide are the azide, p nitrophenylester, 2,4,5trichlorophenylester, the asymmetrical anhydrides and the yreactionproduct of the acid with dicyclohexylcarbodiimide.

The nonapeptide of the invention has a strong blood pressure loweringeffect; this is stronger than that of L-pyroglutamyl-L-prolyl-L-seryl-L-lysyl L aspartyl L-alanyl-L-'phenylalanyl-L-isoleucyl-glycyl L leucyl L- methionine-amide,a hendecapeptide. This last mentioned fact is surprising since thecompound of the invention contains two amino acid radicals less thansaid hendecapeptide. The compound of the invention thus has a propertywhich is useful in antihypertensives for the treatment of high bloodpressure and for controlled blood pressure reduction, e.g. in surgery.

The vascular activity of the nonapeptide of the invention is useful invasodilators for the treatment of circulatory disorders and spasms ofthe vascular system.

An alternative or additional possible use for the nonapeptide of theinvention is as an agent for improving blood circulation in coronaryvessels in the treatment of coronary spasms, attacks of angina pectorisand infarcts and as agent for stopping vaso spasms in the circulary areaof lthe brain vessels; or as agent for promoting the passage ofmedicaments from the blood stream to the tissues.

If the nonapeptide of the invention is used as a therapeutic agent itmay be in the form of a pharmaceutical preparation containing the saidcompound in admixture with an organic or inorganic carrier which issuitable for parenteral application. The pharmaceutical preparation mayalso contain one or more other therapeutically valuable substances.

It should be noted that the nonapeptide may be administered in the formof a depot preparation.

The invention thus also includes pharmaceutical preparations containing,in addition to an inert physiologically acceptable carrier, thenonapeptide of the invention or an acid addition salt thereof.

There is a large number of possible methods of synthesizing thenonapeptide of the present invention; one advantageous method may 'beeffected as follows: N-carbobenzoxy-L-phenylalanine p nitrophenylesteris condensed with L-isoleucine-methylester, the carbobenzoxy radical issplit off and the resulting dipeptide ester is condensed withN-carbo-tert-butoxy-L-alanine. The resulting tripeptide ester,N-carbo-tert-butoxyl-L-alanyl-L phenylalanyl-L-isoleucine-methylester,is converted into the azide via the hydrazide (see FIGURE I of theaccompanying drawings). The azide is condensed with glycyl-L-leucyl-L-methionine-amide, which is obtained by condensation ofN-trityl-glycyl-L-leucine with L-methionine amide 4and splitting ott thetrityl radical. The resulting hexapeptide amide is convertedV intoL-alanyl-L-phenylalanyl-L-isoleucyl-glycyl-L-leucyl-L-methionine-arnideby treatment with triiluoroacetic acid (see FIGURE II :of the drawings).N- carbo-tert-butoxy-L-proline is condensed with L-serinernethylesterand the resulting N-carbo-tert-butoxy-L-prolyl-L-serine-methylester isconverted via the hydrazide into the azide which is condensed withe-N-carbo-tertbutoxy-L-lysine-methylester. The resultingN-carbo-tertbutoxy-L-prolyl-L-seryl-e-N-carbo-tert-butoxy-Llysinemethylester (see FIGURE III of the drawings) is converted via thehydrazide into the azide which is condensed with the above mentionedhexapeptide. Treatment of the protected nonapeptide with triuoroaceticacid yields the biologically active nona'peptide (see FIGURE IV of thedrawings) which, if desired, may be converted into an acid `additionsalt.

In the following example, which illustrates the invention, alltemperatures are indicated in degrees centigrade. Procedure 1 describesthe production of N-carbo-tertbutoxy Lalanyl-L-phenylalany-l-L-isoleucine-methylester (cf. 'FIGURE I),procedure 2 the production of N carbo tert butoxy -L alanyl Lphenylalanyl- L-isoleu-cine-hydrazide, procedure 3 the produc-tion of L-alanyl L phenylalanyl L isoleucyl-glycyl-L-leucyl- Lmethionine-amide,procedure 4 the production of N- carbo tert butoXy L prolyl L seryl e Ncarbotert-butoXy-L-lysine-methylester and procedure 5 the production ofL-prolyl-L-seryl-L-lysyl-L-alanyl-L-phenylalanyl L isoleucyl glycyl L-leucyl L methionineamide.

EXAM1 LE Procedure 1: CTB-Ala-Phe-Ile-OCHB (cf. FIG. I)

168 g. of CBO-Phe-ONP and 58 g. of H-Ile-OCHE are dissolved in 1000 cc.of chloroform, left to stand over night at 20, washed With Water, dilutehydroch-loric acid and aqueous sodium bicarbonate, dried over sodiumsulfate, evaporated in a vacuum land ethyl ether added to Procedure 4:CTB-Pr0-Ser-Lys(CTB)-OCH3 (cf. FIG. Ill) 86 g. of CTB-Pro-OH, 62 g. ofH-'Ser-OCH3-HC1, 56 ml. of triethylamine and 86 g. ofdicyclohexylcarbodiimide are dissolved at 0 in 600 cc. of chloroform,left to stand over night at 0, filtered, evaporated in a vacuum, ethylether added, iltered and petroleum ether added to the solution. 102 `g.of CTB-Pro-Ser-OCH3 are obtained, which are dis-solved in 1100 cc. ofmethanol and 220 cc. of hydrazine hydrate at 0. After 3 days `at 20 itisevaporated in a vacuum, dissolved in methanol and ethyl ether andpetroleum ether added. 79 g. of CTB-Prothe residue. 130 g. ofCBO-Phe-Ile-OCH3 (melting point e 106) crystallize and are dissolved in1100 cc..of a 3.5 N solution of hydrogen bromide in glacial acetic acid.After one hour at 20 it is evaporated in a vacuum, ethyl ether added,the crystallized product dissolved in 600 cc. of chloroform `and 43 cc.of triethylamine, 65 g. of dicyclohexylcarbodiimide and 59 g. ofCTB-Ala-OH added. lt l.is left to stand over night at 0, filtered, thesolution Washed with Water, dilute hydrochloric acid and aqueous sodiumbicarbonate, -dried over sodium sulfate, evaporated in a vacuum andpetroleum ether added to the residue. 105 g. of CTB-Ala-Phe-Ile-OCH3crystallize (melting point 50; [o]D2t=-20 in 95% a-cetic acid).

Procedure 2: CTB-Ala-Phe-lle-NH-NHZ (Cf. FIG. I)

105 g. of CTB-Ala-Phe-Ile-OCH3 and 200 g. of hydrazine hydrate aredissolved in 1000 cc, of methanol. After 4 days at 20, it is evaporatedin `a Vacuum, the residue dissolved in 300 cc. of Water, brought to a pHof 7.5 with 4 N.hydrochloric acid, cooled to 0 and ltered. 55 g. ofCTB-Ala-Phe-Ile-NH-NH2 are obtained (melting point 250 withdecomposition) (after previous heating above 100) []D21=-23 indimethylformamide.

Procedure 3: H-Ala-Pze-IZe-GZy-Leu-Met-NHZ (cf. FIG. 11)

129 g. of Tri-Gly-Leu-OH, 4-5 yg. of H-Met-NH2 and 65 g. ofdicyclohexylcarbodiimide are dissolved in 1300 cc. of methylenechloride, lef-t to stand over night at 0, `-ltered, the solution washedwith dilute hydrochloric acid and aqueous sodium bicarbonate, dried oversodium sulfate,.evaporated in a vacuum and ethyl ether added 112 g. ofTri-Gly-Leu-Met-NHZ are obtained (melting point 212; {a]D21=-4 indimethylformamide) and are left to stand in .a mixture of 600 cc. ofglycial acetic acid and 600 cc. of water for 20 minutes at 90, thencooled to 20 and ltered. The solution is evaporated in a vacuum, theresidue Washed with ethyl ether land crystallized from methanol-ether.62 g. of H-Gly- `Leu-Met-NHZ acetate Iare obtained (melting point 130with decomposition; [a]D21-=35 in 95% acetic acid) `and a solution addedwhich is produced by dissolving 72 g. of CTB-Ala-Phe-Ile-NH-NHZ in 300cc. of 2 N hydrochloric acid and 900 cc. of dimethylformamide at 5 andadding 85 cc. of 2 N sodium nitrite and after 5 `minutes 85 cc. oftriethylamine and 600 cc. of dimethylformamide. It vis lett to standover night at 0, evaporated in `a vacuum, the residue Washed With cold,dilute hydrochloric acid, dissolved in tetrahydrofuran and precipitatedby the addition of Water. 75 g. of CTB-Ala- Ser-NHNH2 are obtained(melting point 80; [MDW: 75 in 95% acetic acid), which are dissolved in750 cc. of N hydrochloric acid at -5. 270 cc. of N sodium nitrite areyadded and after 10 minutes solid sodium carbonate and the resultingazide is extracted with ethyl acetate at 5. The organic solution isdried With sodium sulfate and 71 g. of H-Lys(CT-B)OCH3 are added. After10 hours at 0 and 3 days at 20 it is Washed with dilute hydrochloricacid and aqueous sodium bicarbonate, driedA over sodium sulfate,evaporated in a vacuum, the residue dissolved in ethyl ether `andpetroleum ether added. 95 g. of CTB-Pro-Ser-Lys(CTB)-OCH3 are obtained(melting point 60 with decomposition; [a]D22=-56 in 95% acetic acid).

Procedure 5 H -Pro-Ser-Lys-A la-Phe-I Ie-Gly-Leu-Met- NH, (Cf. FIG. 1V)

g. of CTBPro-Ser-Lys(CTB)-OCH3 are dissolved in 750 cc. of methanol, 200cc. of hydrazine hydrate added, left to stand over night .at 20,evaporated in a vacuum, dissolved in 900 cc. of methanol and 5000 cc.ot' ethyl ether added- 59 g. of CTB-Pro-Ser-Lys(CTB)- DIH-NH2crystallize (melting point 160; [eh21:- 58 in 95% acetic acid), whichare dissolved in 440 cc. of N lhydrochloric acid and 880 cc. ofdimethylformamide; 60 cc. of 2 N sodium nitrite are added at -5 andafter 5 minutes 82 cc. of triethylamine and 57 g. ofH-Ala-'Phe-Ile-Gly-Leu-Met-NH2. It is left to stand overnight at 0,evaporated in a vacuum and the `residue Washed with ethyl ether anddilute acetic acid. 75 g. of nonapeptide are obtained (melting point 260with decomposition; [ot]D20=-50 in 95% acetic acid), which are dissolvedin 2000 cc. of triuoroacetic acid. It is left to stand for one hour lat25, evaporated in a vacuum, the

residue washed with 1000 cc. of Water which is brought to a pH of 6.5 bythe addit-ion of sodium bicarbonate. 57 g. ofH-Pro-Ser-Lys-Ala-Phe-Ile-Gly-Leu-Met-NH2 are obtained in the form of apowder which is ditlicultly soluble in neutral Water. ([a]D29=-73 in 95%triuoroacetic acid. Melting point 220 with decomposition). By means ofacid hydrolysis in an atmosphere of nitrogen it yields proline, serine,lysine, alanine, phenylalanine, isoleucine, glycine leucine methionineand ammonia in equimolecular quantities. The free peptide may bedissolved in Water by the addition of an equivalent of an acid. Thecorresponding salt is obtained by evaporation.

What lis claimed is:

1. A compound selected from the group consisting of L proly-lL-seryl-L-lysyl-L-alanyl-L-phenylalany1-L-isoleucyl-glycyl-L-leucyl-L-methionine amide and its pharmaceuticallyacceptable acid addition salts.

2. L prolyl L seryl L lysyl L alanyl L- phenylalanyl Lisoleucy-l-glycyl-L-leucyl-L-methionine amide.

(References on. following page) 45 6 References Cited Sandrin andBoissonnus: Helv. Chim. Acta. 47, 1294- UNITED STATES PATENTS 1307 and1332 (1964)- 2,766,225 10/1956 Kenner 260 -112.5 Schrorer and Lubke: ThePeptldes, v01. II, New York,

l Acadermc Press, 1966, pp. 1127456. 2,786,049 3/1957 Lundgren 260-112.55 l 0 3,198,705 8/1965 Cummings 16,7 65 Stuermer et a Experlentra 20,303-3` 6 (1964). 3,203,860 8/ 1965 Wu et a1. 167-65 3,268,502 8/1966Lubke et a1. 26o-11.2.5 LEWIS GOTTS Plmary Exammer 3,272,790 9/ 1966Bernardi et a1. 260-112-5 JULIAN S. LEVITT, Examiner.

OTHER REFERENCES 10 LEROY B. RANDALL, MELVYN M. KASSENOFF,

Bernardi et a1.: Experientia 20, 306-309 (1964). Assistant Examiners.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OFL-PROLYL-L-SERYL-L-LYSYL-L-ALANYL-L-PHENYLALANYL-LISOLEUCYL-GLYCYL-L-LEUCYL-L-METHIONINE AMIDE AND ITS PHARMACEUTICALLY ACCEPTABLE ACID ADDITIONSALTS.